Adaption of the Cytokinesis-Block Micronucleus Cytome Assay for Improved Triage Biodosimetry.

The purpose of this work was to adapt a more advanced form of the cytokinesis-block micronucleus (CBMN) cytome assay for triage biodosimetry in the event of a mass casualty radiation incident. We modified scoring procedures for the CBMN cytome assay to optimize field deployability, dose range, accuracy, speed, economy, simplicity and stability. Peripheral blood of 20 donors was irradiated in vitro (0-6 Gy X ray, maximum photon energy 240 keV) and processed for CBMN. Initially, we assessed two manual scoring strategies for accuracy: 1. Conventional scoring, comprised of micronucleus (MN) frequency per 1,000 binucleated (BN) cells (MN/1,000 BN cells); and 2. Evaluation of 1,000, 2,000 and 3,000 cells in total and different cellular subsets based on MN formation and proliferation (e.g., BN cells with and without MN, mononucleated cells). We used linear and logistic regression models to identify the cellular subsets related closest to dose with the best discrimination ability among different doses/dose categories. We validated the most promising subsets and their combinations with 16 blind samples covering a dose range of 0-8.3 Gy. Linear dose-response relationships comparable to the conventional CBMN assay (r(2) = 0.86) were found for BN cells with MN (r(2) = 0.84) and BN cells without MN (r(2) = 0.84). Models of combined cell counts (CCC) of BN cells with and without MN (BN(+MN) and BN(-MN)) with mononucleated cells (Mono) improved this relationship (r(2) = 0.92). Conventional CBMN discriminated dose categories up to 3 Gy with a concordance between 0.96-1.0 upon scoring 1,000 total cells. In 1,000 BN cells, concordances were observed for conventional CBMN up to 4 Gy as well as BN(+MN) or BN(-MN) (about 0.85). At doses of 4-6 Gy, the concordance of conventional CBMN, BN(+MN) and BN(-MN) declined (about 0.55). We found about 20% higher concordance and more precise dose estimates of irradiated and blinded samples for CCC (Mono + BN(+MN)) after scoring 1,000 total cells. Blinded sample analysis revealed that the mean absolute difference (MAD) of dose estimates and the number of dose estimates outside the ±0.5 Gy interval based on CCC (Mono + BN(+MN)) was comparable to conventional CBMN for doses ≤4 Gy when scoring 3,000 total cells or more. At doses >4-8.3 Gy, the MAD of CCC (Mono + BN(+MN)) declined to half of the MADs observed for conventional CBMN, suggesting that the combined cell counts approach improved the discrimination ability. Conventional CBMN at 1,000 total-cell counts performed as efficiently as counting 1,000 BN cells. Discriminating and counting only BN cells with and without MN after 1,000 BN cells at ≤4 Gy revealed performances similar to conventional CBMN. After 3,000 total cells were scored, CCC (Mono + BN(+MN)) was superior to conventional CBMN at >4 Gy up to about 8 Gy. Our modification of CBMN evaluations saved time compared to the widely established semiautomated MN scoring and extended the dose range up to approximately 6 Gy for triage biodosimetry.

Contralateral biopsies in patients with testicular germ cell tumours: patterns of care in Germany and recent data regarding prevalence and treatment of testicular intra-epithelial neoplasia.

The precursor of testicular germ cell tumours (GCTs), called testicular intra-epithelial neoplasia (TIN/CIS), is safely diagnosed immunohistologically. Testicular biopsy provides a valuable tool for early detection of GCTs in risk groups. Although this knowledge is undisputed, testicular biopsies are utilized poorly. The patterns of care regarding the use of biopsies remain unknown. Uncertainty exists about the prevalence and specific treatment of TIN/CIS. We asked clinical urologists in Germany whether or not they employed contralateral biopsies in GCT patients. We evaluated the prevalence of contralateral TIN/CIS in a retrospective analysis of 780 consecutive GCT patients. All had contralateral double biopsies. Discordance of TIN/CIS findings among biopsy pairs as well as age, histology of the primary tumour and clinical stage was noted. Evaluation of data comprised descriptive statistical methods. To evaluate treatment options for TIN/CIS, we performed a literature search. 52.1% of German urologists always perform the biopsy, 17% do it mostly, 27.3% in select cases, 3.5% never. Curiously, there was a geographic north-south gradient regarding biopsy use. Contralateral TIN/CIS was found in 5%. The median ages of patients with TIN/CIS and those without were 31.8 and 34.9 years respectively (p = 0.02). The discordance rate among biopsy pairs was of 33%. Two-site biopsies provide a 17% gain in diagnostic sensitivity. Local radiotherapy with 20 Gy is the safest treatment of TIN/CIS failing in 2%. Chemotherapy has significantly lower efficacy. Contralateral testicular biopsies in GCT patients are well accepted among German urologists. The prevalence of contralateral TIN/CIS found in this series is in accordance with previous reports. Double biopsies should be the diagnostic standard because of their diagnostic superiority. Local radiotherapy with 20 Gy is the safest way of eradicating TIN/CIS. Failures occur in only 2%, usually many years after irradiation. Cisplatin-based chemotherapy is dose dependent and less effective.

Discriminating metastasised from non-metastasised seminoma based on transcriptional changes in primary tumours using NGS.

BACKGROUND: We aimed to better discriminate (occult) metastasised from non-metastasised seminoma based on transcriptional changes of small RNAs in the primary tumour. METHODS: Total RNAs including small RNAs were isolated from five testicular tumours of each, lymphogenic, occult and non-metastasised patients. Next-generation sequencing (SOLID, Life Technologies) was used to examine transcriptional changes. Small RNAs showing ⩾50 reads and a significant ⩾2-fold difference using non-metastasised tumours as the reference group were examined in univariate logistic regression analysis and combinations of two small RNAs were further examined using support vector machines. RESULTS: On average, 1.3 × 10(7), 1.4 × 10(7) and 1.7 × 10(7) small RNA reads were detectable in non-metastasised, occult and lymphogenic metastasised seminoma, respectively, of which 30-32% remained after trimming. Between 59 and 68% represented annotated reads and between 8.6 and 11% were annotated small RNA tags. Of them, 137 small RNAs showed>50 reads and a two-fold difference to the reference. In univariate analysis, 32-38 small RNAs significantly discriminated lymphogenic/occult from non-metastasised seminoma, and among these different comparisons, it were the same small RNAs in 51-88%. Many combinations of two of these small RNAs allowed a complete discrimination of metastasised from non-metastasised seminoma irrespective of the metastasis subtype. CONCLUSIONS: Metastasised and non-metastasised seminoma can be completely discriminated with a combination of two small RNAs.

Prediction of metastatic status in non-seminomatous testicular cancer.

PURPOSE: To examine the significance of 90 biomarkers for predicting metastatic status in non-seminomatous germ cell tumors (NSGCT). By predicting metastatic status, it may be possible to eliminate unnecessary therapeutic or diagnostic efforts. MATERIALS AND METHODS: We investigated 552 males who were diagnosed with non-metastatic (n = 273) and metastatic (n = 279) NSGCT between 2000 and 2011. The sample included cancers of different histologies: embryonal cell carcinoma (n = 131), teratoma (n = 55), and mixed histology (n = 366). We collected and analyzed more than 90 parameters via logistic regression: demographic characteristics, medical history, histopathological parameters, and levels of tumor markers and hormones. RESULTS: Testis histology (p = 0.004), clinical symptoms (p = 0.0005), tumor length (p = 0.005), infiltration of the rete testis (p = 0.008), invasion of lymphatic (pL1) and blood vessels (pV1) (p < 0.0001), and levels of enzymes such as LDH, ßHCG, AFP, and FSH (p values as small as <0.0001) were associated with metastatic status. With one model, we identified 14 out of 76 (18.4 %) metastatic NSGCT cases with 93-100 % certainty (positive predictive value) at 99 % specificity by the peripheral blood levels of LDH (day of operation) in combination with FSH measurements (1 day after operation). A second model included pV, tumor length, and FSH (1 day after operation). It identified 25 out of 90 (27.8 %) non-metastatic NSGCT with approximately 90 % certainty (negative predictive value) at 94-98 % sensitivity. CONCLUSIONS: No single parameter was able to discriminate metastatic from non-metastatic NSGCT, but combinations of parameters in two predictive models accurately identified the metastatic status in 23 % of the cases in our sample.

The search for biomarkers of metastatic seminoma.

PURPOSE: We screened 90 potential parameters as biomarkers of metastatic seminoma to facilitate detection and eliminate unnecessary therapeutic or diagnostic efforts. MATERIALS AND METHODS: A total of 527 men with pure seminoma (diagnosed 2000 to 2011) were followed during therapy. More than 90 demographic/anamnestic (eg age, height, weight) histopathological parameters (testicular/tumor size, testicular intraepithelial neoplasia) and levels of tumor markers (eg α-fetoprotein, ß-human chorionic gonadotropin, lactate dehydrogenase) in peripheral blood and testicular vein were collected for analysis via logistic regression. Previously described risk factors (tumors larger than 4 cm, infiltration of rete testis) were assessed separately. RESULTS: Established parameters such as tumor length (p = 0.0003), involvement of lymphatic (p <0.0001) or vascular channels (p = 0.0009), extent of primary tumor (p <0.0001) and infiltration of the tunica albuginea (p = 0.02) as well as new biomarkers such as absence of testicular intraepithelial neoplasia in tumor bearing testis (p = 0.03), testicular volume (p = 0.04) and tumor volume (p = 0.02) showed a significant association with metastatic disease. This association was also true of lactate dehydrogenase, human chorionic gonadotropin and α-fetoprotein (p <0.0001 at maximum). However, the discriminatory capacity of these biomarkers (concordance or ROC area) did not exceed 65% when examined alone or in combination, and higher values (up to 80%) were detected for enzyme levels. A subset of metastatic seminoma (2% to 27%) was detectable with high accuracy (positive predictive value 92% to 100%) based on enzyme measurements (p <0.0006). CONCLUSIONS: New biomarkers of metastatic seminoma were identified and previously described risk factors were validated. Further prospective studies of these novel parameters are warranted to verify our findings and to explore a potential use for detecting occult metastases.

[Value of targeted treatment for testicular cancer: from molecular approaches to clinical possibilities]. / Der Stellenwert der Targeted-Therapie beim Hodentumor: Von molekularen Ansätzen zu klinischen Möglichkeiten.

Due to the introduction of tyrosine kinase-inhibitors in the treatment of metastatic renal cell cancer, targeted therapy raises hopes for other urological tumors as well. Even if excellent cure rates, achieved by standardization of diagnosis und therapy, have made testicular cancer a curable disease, up to 6% of young patients still die from tumors refractory to therapy. The quality of life of patients in advanced stages needing aggressive treatment should be improved by new therapies with reduced side effects. The role of tyrosine kinase inhibitors and angiogenesis inhibitors as well as intervention in the cell cycle and induction of apoptosis are discussed.